Materials

The materials used for cell cultures and characterization are listed in Additional file 1: Table S1.

Cell sources

After getting informed consent, PB was drawn from three healthy O, Rh D-positive donors. CB was collected from three healthy newborn babies at the Department of Obstetrics and Gynecology at Severance Hospital in Seoul, South Korea. Three normal frozen BM aspirate samples were acquired from the Department of Internal Medicine at the same hospital. The study protocol was reviewed and approved by the Institutional Review Board of Severance Hospital (IRB No. 4-2018-0890).

Production of hiPSCsIsolation of mononuclear cells

About 15–20 mL PB and BM aspirates and 100 mL umbilical CB were collected in a tube containing sodium heparin anticoagulant (BD Biosciences, Oxford, UK). Mononuclear cells (MNCs) were purified using 10 mL Ficoll-Paque (GE Healthcare, Uppsala, Sweden). Viable MNCs were counted using the trypan blue exclusion method [25].

Erythroid progenitor expansion

Viable MNCs were resuspended and plated at a density of 1 × 106 cells/mL on erythroid expansion media composed of basal medium and erythroid cytokines. The composition of the basal medium was 150 μg/mL transferrin, 50 μg/mL insulin, 90 ng/mL ferrous nitrate, and 160 μM monothioglycerol in Stemline II media (all from Sigma-Aldrich, Gillingham, UK). The erythroid expansion medium was prepared by adding 10 μg/mL hydrocortisone (HC) (Sigma-Aldrich), 10 μg/mL stem cell factor (SCF) (Sigma-Aldrich), 10 μg/mL erythropoietin (EPO) (Stem Cell Technologies, Vancouver, Canada), and 1 μg/mL interleukin-3 (IL-3) (Peprotech EC Ltd., London, UK) to the basal medium. MNCs were suspended at a density of 1 × 107 cells/mL in 10 mL erythroid expansion medium in a 25T flask (Thermo Fisher Scientific, Waltham, MA, USA) for three days in a 37 °C incubator in a humidified environment containing 5% CO2. On day 3, both adherent and non-adherent cells were collected into a 15-mL conical tube and centrifuged at 400× g for 5 min. The pellet was resuspended at a density of 1 × 106 cells/mL in fresh erythroid expansion medium. Starting on day 5, microscopic morphological analysis was performed every day until the population of erythroid progenitor cells accounted for approximately 80% of the MNCs, at which point the cells were ready for transfection [26].

Transfection

Prior to transfection, each well in 6-well Nunc™ Multidishes (Thermo Fisher Scientific) was coated with a mixture of 14.5 μL Matrigel Matrix (Stem Cell Technologies) and 985.5 μL Dulbecco's Modified Eagle Medium (DMEM)/F12 (1X) (Gibco, Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. A total of 1 × 106 expanded erythroid cells were centrifuged at 400× g for 5 min and resuspended in 100 μL Opti-MEM media (Gibco); 2 μL the Epi Episomal Reprogramming Vectors, pCE-hOCT3/4 (OCT4), pCE-hSK (SOX2, KLF4), and pCE-hUL (L-Myc, Lin28); and 2 μL Epi p53 and EBNA vectors, pCE-mP53DD (mp53DD), and pCXB-EBNA1 (EBNA1) (Life Technologies, Frederick, MD, USA). Prepared cells were transferred to Electroporation Cuvettes (Nepa Gene Co., Ltd., Chiba, Japan) and loaded into a NEPA21 Super Electroporator (Nepa Gene Co., Ltd.). Transfection was performed as per the manufacturer’s instructions. The transfected cells were transferred to 6 mL of erythroid expansion medium, and 2 mL of the mixed product was plated on a 6-well plate pre-coated with Matrigel at a density of 3.3 × 105 cells/well. On the second and fifth days after transfection, each well was supplemented with 1 mL erythroid expansion medium and 1 mL ReproTeSR (Stem Cell Technologies), respectively. Beginning on post-transfection day 7, complete medium changes were performed with 2 mL ReproTeSR every day, and the morphology of colonies was closely monitored until the hiPSC-like colonies appeared [26, 27].

Maintenance of hiPSCs

The produced hiPSCs were maintained on the plates with 2 mL mTeSR Plus Basal media (Stem Cell Technologies) in an incubator containing 5% CO2 at 37 °C and cultured daily with fresh mTeSR until the cells reached 80–90% confluence. Within five to seven days, hiPSC colonies were ready for passaging. For newly reprogrammed hiPSCs up to approximately passage five, colonies were manually passaged using a loop made from a heated glass Pasteur pipette to select only preferred colonies of undifferentiated hiPSCs. For the subsequent passages, hiPSCs were enzymatically passaged using ReLeSR (Stem Cell Technologies). The medium was changed daily. Cultured hiPSCs between passages 10 and 15 were used for erythroid differentiation in the study [26].

Differentiation into hematopoietic stem cell and erythroid cell lineages

We used a modified stepwise protocol cited in other studies [19, 26]. The diagram depicting the hematopoietic and erythroid differentiation of hiPSCs is illustrated in Fig. 1.

Fig. 1

Diagrammatic representation of the hematopoietic and erythroid differentiation of hiPSCs. Abbreviations: KOSR, Knock Out Serum Replacement; BMP4, bone morphogenetic proteins 4; VEGF, vascular endothelial growth factor; b-FGF, fibroblast growth factor-basic; SCF, stem cell factor; IGF2, insulin-like growth factor 2; IBMX, 3-isobutyl-1-methylxanthine; SR1, StemReagenin1; HC, hydrocortisone; IL-3, interleukin-3; EPO, erythropoietin

Mesodermal differentiation

On day 0 of differentiation, a new medium composed of Roswell Park Memorial Institute (RPMI) (Gibco) with 10% KnockOut Serum Replacement (KOSR) (Gibco), 100 ng/mL bone morphogenetic proteins 4 (BMP4), 3 μM/mL IWP2, 50 ng/mL vascular endothelial growth factor (VEGF), and 10 ng/mL Y-27632 was prepared. The existing medium of hiPSC colonies was removed, and 2 mL Dulbecco’s phosphate-buffered saline (DPBS) was added to each well. After removing the DPBS, 1 mL ReLeSR was added and suctioned immediately. The plates were incubated in an incubator containing 5% CO2 for 3 min. Colonies of hiPSC were dissociated into small clumps containing 30–50 cells. After confirming the size of the clumps under a stereo microscope, the cells were resuspended in the prepared media at a density of 3–5 × 106 cells/well on Costar 6-well Clear Flat Bottom Ultra-low Attachment Multiple Well Plates (Corning Life Sciences, Durham, NC, USA). On day 1, 10 ng/mL fibroblast growth factor-basic (b-FGF) was added to each well and maintained for one day. On day 3, a complete medium change using RPMI with 10% KOSR, 100 ng/mL BMP4, 3 μM/mL IWP2, 15 ng/mL VEGF, 10 ng/mL b-FGF, and 10 ng/mL Y-27632 was performed, and the cells were maintained for two more days. For complete medium changes, cell suspensions were centrifuged at 400× g for 5 min, and the cells were reseeded in fresh media.

Hematopoietic commitment and amplification

On day 6, half of the medium was exchanged for Stemline II hematopoietic stem cell expansion medium (Sigma-Aldrich), 20 ng/mL BMP4, 30 ng/mL VEGF, 10 ng/mL b-FGF, 30 ng/mL SCF, 10 ng/mL insulin-like growth factor 2 (IGF2), 5 μg/mL heparin, and 50 μM 3-isobutyl-1-methylxanthine (IBMX). On day 7, all cells were collected and centrifuged at 400× g for 5 min. The pellet was dissociated with medium containing Stemline II hematopoietic stem cell expansion medium (Sigma-Aldrich), 20 ng/mL BMP4, 30 ng/mL VEGF, 10 ng/mL b-FGF, 30 ng/mL SCF, 10 ng/mL IGF2, 5 μg/mL heparin, and 50 μM IBMX. The cells were re-plated in a 25T flask (Thermo Fisher Scientific) and maintained for three days. On day 10, a complete medium change was performed with the same freshly mixed medium used on day 7 with the addition of 1 μM StemReagenin1 (SR1), and the cells were cultured for three more days.

Erythroid differentiation and maturation

On day 13, the cells were re-plated in a basal medium with the following cytokines: 1 × 10−6 M, 100 ng/mL SCF, 10 ng/mL IL-3, and 6 IU/mL EPO. On day 20, a basal medium mixed with 50 ng/mL SCF, 10 ng/mL IL-3, and 6 IU/mL EPO was used. On days 13–27, a complete medium change was performed every three to four days. Starting on day 27, the cells were subjected to a complete medium change with basal media containing 6 IU/mL EPO and 0.10% F68 every three to four days until the cells fully matured into reticulocytes.

Characterization of differentiated cellsMorphological analysis

Cells (1 × 105 per slide) were centrifuged and immobilized onto a glass microscope slide using a Cytospin 5 cytocentrifuge (Thermo Fisher Scientific) at 700 rpm for 7 min. The slides were stained with Wright–Giemsa dye (Sigma-Aldrich), observed under a BX53 light microscope (Olympus, Tokyo, Japan), and imaged with a DP70 camera (Olympus) [26, 27].

Flow cytometric analysis

To investigate the expression of the hiPSC pluripotency markers, SSEA4 and TRA-1-60, hiPSCs were dissociated using Gentle Cell Dissociation Reagent (Gibco) and centrifuged at 400× g for 5 min. The pellet was mixed with autoMACS Running Buffer (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany) and aliquoted into cryotubes at a density of 1 × 105 cells/200 μL with 0.5 M ethylenediaminetetraacetic acid (pH 8.0, 1:90 DPBS). Fluorochrome-conjugated antibodies (10 μL/105 cells) were added to the cryotubes and incubated in the dark for 1 h at room temperature. Unbound antibodies were washed with 1000 μL Fluorescence-Activated Cell Sorting (FACS) buffer. After centrifugation at 400× g for 5 min, the pellet was resuspended in 500 μL 4% paraformaldehyde (Tech&Innovation, Gyeonggi-do, South Korea) for fixation.

To evaluate the hematopoietic and erythroid characteristics of differentiated cells on days 0, 13, 20, 27, 31, and 34, flow cytometric analysis was performed using antibodies against CD34-PE, CD43-APC, CD235a-PE, and CD71-APC (all from BD Biosciences, Oxford, UK). Cells were centrifuged at 400× g for 5 min, and the pellet was dissolved in 400 μL FACS buffer. The subsequent steps were the same as those mentioned above. Non-specific immunoglobulin isotype controls of the corresponding class served as a negative control. Compensation beads were used to modify compensation matrices.

Stained samples were measured in a BD Verse flow cytometer (BD Biosciences). FlowJo software version 10.2 (LLC, Ashland, OR, USA) was used to analyze the data [26,27,28].

Immunofluorescence assay

Reprogrammed cells were gently washed with DPBS and fixed in 4% paraformaldehyde at room temperature for 20 min. The cells were washed again with 0.05% Tween-20 (Sigma-Aldrich) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) at room temperature for 15 min. After washing, they were blocked with 4% Donkey Serum at 4 °C overnight and wrapped in parafilm. Primary antibodies, SSEA4, OCT4, SOX2, TRA-1-60, and NANOG, were added to the cells and then, incubated at 4 °C overnight. The secondary antibodies, Alexa Fluor 594 anti-rabbit or Alexa Fluor 488 anti-rabbit antibodies (Life Technologies, Eugene, OR, USA), were supplemented after washing with 0.05% Tween-20. The samples were incubated at 4 °C overnight in the dark and stained with UltraCruz Aqueous Mounting Medium with DAPI (Santa Cruz Biotechnology, Dallas, TX, USA) for observation.

A CKX53 fluorescence microscope (Olympus, Tokyo, Japan) with a U-RFL-T fluorescence lamp (Olympus) was used to visualize the cells. Image analysis and colocalization studies were carried out using the Ocular Image Acquisition Software, version 2.0.1.496 (Digital Optics Limited, Auckland, New Zealand) [26].

Karyotyping

hiPSCs were fixed and examined using standard G-banding analysis for karyotyping, which was performed by GenDX (Seoul, South Korea) utilizing a GTG-banding technique [29].

Quantitative real-time polymerase chain reaction (qRT-PCR) for detection of various markers

For pluripotent stem cell markers, hiPSCs were grown for four to five days and cultured for analysis. For the three germ layer markers, hiPSCs were maintained on a 10-cm plate coated with 0.1% gelatin solution (GenDEPOT, Katy, TX, USA) in 10 mL germ layer differentiation media composed of DMEM (Gibco) and 10% Fetal Bovine Serum (Gibco). The cells were grown for two weeks with complete medium changes every two to three days.

Total RNA from hiPSC samples was extracted using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Complementary DNA was generated using the iScript cDNA Synthesis Kit (BIO-RAD, Hercules, CA, USA). qRT-PCR was performed using TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA) and analyzed using the Step One Plus (Applied Biosystems). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used to normalize data, and relative expression was calculated using the ΔΔCT method. The statistical significance of the differences between samples was analyzed using a two-way analysis of variance (ANOVA) in GraphPad Prism 9, version 9.2.0 (GraphPad Software, San Diego, CA, USA).

The following qPCR TaqMan probes (Applied Biosystems) were used: POU5F1(OCT4) Hs04260367_gH_FAM; NANOG H02387400_g1_FAM; SOX2 Hs01053049_s1_FAM; KLF4 Hs00358836_m1_FAM; c-MYC Hs00153408_m1_FAM; Brachyury Hs00610080_m1_FAM; Sox17 Hs00751752_s1_FAM; Nestin Hs04187831_g1_FAM; and GAPDH Hs02758991_g1_VIC [21, 26, 30, 31].

Hemoglobin composition analysis using quantitative real-time PCR

On day 31, total RNA from hiPSC-derived erythroid cells was extracted using an RNeasy Plus Mini Kit (Qiagen). Complementary DNA was prepared using an iScript cDNA Synthesis Kit (BIO-RAD) and PowerSYBR Green PCR Master Mix (Life Technologies, Ltd., Warrington, UK). qRT-PCR was performed using Step One Plus (Thermo Fisher Scientific) as per the manufacturer's instructions. GAPDH was used as the house keeping gene for normalizing sample quantities. Relative expression was calculated using the ΔΔCT method. Primers were synthesized by Bioneer (Seoul, South Korea) (Additional file 1: Table S2) [16, 30].

Oxygen-binding capacity analysis

A Hemox-Analyzer (TCS, Southampton, PA, USA) was used to determine the oxyhemoglobin dissociation curves and P50 values of the samples on day 34. Five milliliters HEMOX solution, 20 μL Additive-A, and 10 μL anti-foaming agent (all from TCS) were added to the sample vial and mixed with the sample. The vial was loaded into the cuvette, and the analysis was performed as per the manufacturer’s instructions. The data obtained from hiPSC-differentiated erythroid cells were compared to those obtained from freshly drawn normal PB [21, 27, 32].

Scanning electron microscopy

On day 34, hiPSC-derived erythroid cells were fixed in Karnovsky’s fixative, which was composed of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer at a pH of 7.4 (all from Merck, Darmstadt, Germany), for 24 h and washed with 0.1 M phosphate buffer. The prepared samples were post-fixed with 1% osmium tetroxide (Polysciences, Washington, PA, USA) for 2 h and dehydrated through incubation in a series of ethanol solutions (50–100%). Critical point-drying was performed using an EM CPD300 (Leica Microsystems, Wetzlar, Germany) for 1–2 h. The specimens were mounted using a Stemi 305 (Zeiss, Jena, Germany) and coated with platinum using a Leica EM ACE600 ion sputtering system (Leica Microsystems, Wetzlar, Germany). The prepared specimens were observed using a Merlin field emission electron microscope (Zeiss, Jena, Germany) [33].

RNA sequencing

For transcriptomic comparison of hiPSCs, RNA sequencing was carried out on six hiPSC lines (n = 2 for each source), and the genes expressed in CB- and BM-derived hiPSCs were compared to those obtained from PB-derived hiPSCs, which were set as control. To identify changes in transcriptomic expression during differentiation, RNA sequencing was performed on days 13, 20, and 27 (n = 1 for each source), corresponding to three stages of erythroid differentiation, and the transcriptomic profiles on each day were compared to those of their precursor hiPSCs.

Samples of approximately 1 × 107 cells/mL on days 0, 13, 20, and 27 were lysed using 1 mL TRIzol Reagent (Life Technologies) according to the manufacturer’s instructions and stored at − 70 °C until RNA extraction. RNA preparation, sequencing, and analysis were all performed by Macrogen, Inc. (Seoul, South Korea). The quality of isolated RNA samples was evaluated with Tape Station RNA Screen Tape (Agilent, Santa Clara, CA, USA). cDNA libraries were prepared using a TruSeq Stranded mRNA LT sample Prep Kit (Illumina, San Diego, CA, USA), and the transcribed cDNA was sequenced using a NovaSeq 6000 (Illumina).

The raw sequences were quality-checked using FastQC v.0.11.7, and low-quality bases and the adaptor contamination were removed using Trimmomatic v.0.38. Trimmed reads were mapped to the human genome reference, namely either UCSC hg19 or GRCh37, using HISAT2 (Johns Hopkins University, Baltimore, MD, USA). The total mapped read numbers were determined and normalized to detect the number of fragments per kilobase of transcript per million mapped reads and transcripts per kilobase. Differentially expressed gene analysis was performed using these values [34, 35].

Statistics and reproducibility

At least three independent differentiation experiments were performed for each cell line. All statistics were analyzed using GraphPad Prism, version 9.2.0 (GraphPad Software, San Diego, CA, USA). Values were considered statistically significant if the p-value was less than 0.05.